bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Pulse of α-2-macroglobulin and lipocalin-1 in the pregnant uterus of
European polecats (Mustela putorius) at the time of implantation
Heli Lindeberga, Richard J.S. Burchmoreb, and Malcolm W. Kennedyc
a
Natural Resources Institute Finland (Luke), Green Technology, Halolantie 31 A, FIN-71750
Maaninka, Finland.
b
Institute of Infection, Immunity and Inflammation, and Glasgow Polyomics, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Garscube Campus, Glasgow G61 1QH, Scotland,
UK.
c
Institute of Biodiversity, Animal Health and Comparative Medicine, and the Institute of Molecular,
Cell and Systems Biology, Graham Kerr Building, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, Scotland, UK
Author for correspondence: Malcolm Kennedy ([email protected])
Keywords European polecat, ferret, Mustela putorius, uterine secretions, pregnancy, implantation,
proteomics.
Short title: Proteomics of polecat uterine fluids in early pregnancy
Proteomics of polecat uterine fluids in early pregnancy
Page 1
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Abstract
Uterine secretory proteins protect the uterus and conceptuses against infection, facilitate
implantation, control cellular damage resulting from implantation, and supply embryos with
nutrients. The early conceptus of the European polecat (Mustela putorius) grows and develops free
in the uterus until implanting at about 12 days after mating. Using a proteomics approach we found
that the proteins appearing in the uterus leading up to and including the time of implantation
changed dramatically with time. Several of the proteins identified have been found in pregnant uteri
of other placental mammals, such as α1-antitrypsin, serum albumin, lactoferrin, cathepsin L1,
uteroferrin, and ectonucleotide pyrophosphatase. The broad-spectrum proteinase inhibitor α2macroglobulin rose from relatively low abundance initially to dominate the protein profile by the
time of implantation. Its functions may be to limit damage caused by the release of proteinases
during implantation, and to control other processes around the site of implantation. Lipocalin-1 (also
known as tear lipocalin) has not previously been recorded as a uterine secretion in pregnancy, and
also increased substantially in concentration. If polecat lipocalin-1 has similar biochemical properties
to the human form, then it may have a combined function in transporting or scavenging lipids, and
antimicrobial activities. The changes in the uterine secretory proteome of Euroepan polecats may be
similar in those species of mustelid that engage in embryonic diapause, but possibly only following
reactivation of the embryo.
1. Introduction
A conceptus may implant soon after its arrival in the uterus (as in humans and muroid rodents), or
may remain free in the uterus for several weeks before implanting (as in equids) [1]. Intermediate to
these extremes are the conceptuses of many Carnivora and Artiodactyla which implant 10-20 days
after mating. The delay is attributed to time required for mating-induced ovulation, travel through
the fallopian tubes, and a variable period during which the blastocyst develops without close cellular
contact with maternal tissues [1]. In some species of mustelid, ursids, phocids, and roe deer,
implantation is postponed for several months, during which time the conceptus enters obligatory
diapause at the blastocyst stage and must be maintained and protected until reactivation is
permitted by the mother [2-5]. The European polecat, Mustela putorius [6], fits into the
intermediate group, with an embryonic development period (without embryonic diapause) of
approximately 12 days preceding implantation.
Proteomics of polecat uterine fluids in early pregnancy
Page 2
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Proteins secreted into the non-pregnant uteri of eutherian mammals have a range of presumptive
functions such as maintenance of the mucocutaneous surface of the endometrium, antimicrobial
protection, receptivity to sperm and the subsequent conceptus, and nutrition of an embryo. Distinct
differences have been found among mammal groups in the proteins secreted into the uterus before
placentation, although there are commonalities [7-12]. These proteins have been divided mainly into
those involved in nutritional support for the conceptus, or in facilitating and controlling implantation
events. Perhaps the most notable example of the nutritional function is the uterocalin/P19 protein in
horses. This protein appears to deliver essential lipids such as retinol and polyunsaturated fatty acids
across the glycoprotein capsule to the equine conceptus [13-15]. Lipids present a problem in their
transfer from mother to conceptus because they tend to be insoluble, and in some cases susceptible
to oxidation damage unless protected within a protein binding site. Uterocalin is also notable in that
its primary structure is enriched in essential amino acids, which doubles its function as a nutrient
source for the developing embryo [15]. Other examples of lipid carriers in uterine secretions include
a modified form of plasma retinol binding protein, and serum albumin which binds a range of small
molecules, fatty acids in particular [11, 16, 17]. Of those proteins considered to influence
implantation events, the broad-spectrum proteinase inhibitor α-2-macroglobulin (α2M) is secreted
into the pregnant uterus around the time of implantation in several species, and, among other roles,
is thought to limit tissue damage during implantation and to control local inflammatory responses
[7-12, 18, 19].
Overall, there is considerable diversity in the suite of proteins secreted into pre-placentation uteri by
different clades of mammals, as exemplified by equids, artiodactyles and Carnivora, all of which
exhibit distinctive repertoires [7-10, 12]. We investigated the proteins found in uterine secretions of
pregnant European polecats in order to explore the preparatory events that lead up to, and at,
implantation. Analysis of chronological samples indicated that progressive and dramatic changes in
the protein profile occur as the time at which implantation would occur approaches. Some of the
proteins have been found in pre-implantation uterine flushes of other species (notable among which
is α-2-macroglobulin), although not in the same combination or relative concentrations. At least one
protein, lipocalin-1, which we found to peak in abundance at implantation, has previously not been
reported as a uterine secretory protein. Not only does this study demonstrate that mustelids exhibit
a different but overlapping repertoire of implantation-related uterine secretions from other species
groups, but it may also provide a general framework for investigation of what happens during
embryonic quiescence and subsequent reactivation in species that engage in embryonic diapause.
Proteomics of polecat uterine fluids in early pregnancy
Page 3
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
2. Materials and Methods
2.1 Animals and sample collection
The subjects used in this study descended from a population of 70 wild European polecats (Mustela
putorius) captured in the 1970s and interbred with captive 200 imported domestic ferrets
(subspecies Mustela putorius furo). The resulting population forms the basis for all polecats farmed
for the fur industry and research in Finland. The animals were maintained at the Kannus Research
Farm Luova Ltd., in Kannus, Finland. They were held in individual cages measuring 70 x 30 x 38 cm
(length x width x height) with nest boxes measuring 40 x 29 x 32 cm which were accessible to the
females but external to the main cage areas. During the breeding season, the animals were exposed
to outdoor temperatures and light conditions: mean 2°C and 15 hours light in April, 9°C and 16 hours
light in May, 14°C, and 20 hours light in June. The animals were fed according to standards for
breeding farmed polecats in Finland (prepared to provide at least 1150-1250 kcal/kg, 40-50%
protein, 30-32% fats, and 15-25% carbohydrates, supplemented with vitamins and minerals), and
had water provided ad libitum.
The time of oestrus was estimated by the physical appearance of the vulva. Males used for mating
were known to have been fertile the previous year, and matings were visually confirmed (a tie
observed). Animals were randomly selected for sample collection on days 4, 6, 7, 9, 12 and 14 days
after mating, and those sampled on days 4 and 14 were found not to have become pregnant and
thereby acted as mated but non-pregnant controls. Each animal was anesthetized with
medetomidine (Dorbene®; Laboratorios syva s.a., León, Spain) and ketamine (Ketalar®; Pfizer Oy,
Helsinki, Finland), and the uterus removed under sterile surgical conditions. The animals were
subsequently euthanized with T-61 (Intervet International B.V., Boxmeer, the Netherlands) while still
under anesthesia. Once the uterus was removed, the tips of each horn were opened, and the cut
ends cleared of blood. The cervix was closed with forceps, and the uterus flushed with 5 ml sterile
physiological saline through one horn and out the other, with care to avoid blood contamination.
The flush fluid was filter-sterilised and frozen at -20 °C in approximately 2 ml aliquots. Pregnancy
was confirmed by the presence of developing embryos in uterine flushes.
All of the work was approved by the National Animal Experiment Board (Eläinkoelautakunta, ELLA)
of Finland.
2.2 Protein electrophoresis
Proteomics of polecat uterine fluids in early pregnancy
Page 4
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1-dimensional vertical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out using the Invitrogen (Thermo Scientific, Paisley, UK) NuPAGE system using precast 4-12%
gradient acrylamide gels, with β2-mercaptoethanol (25 μl added to 1 ml sample buffer) as reducing
agent when required. Gels were stained for protein using colloidal Coomassie Blue (InstantBlue,
Expedion, Harston, UK) and images of gels were recorded using a Kodak imager. Electronic images
were modified only for adjustment of contrast and brightness. Pre-stained molecular mass/relative
mobility (Mr) standard proteins were obtained from New England Biolabs, Ipswich, MA, USA (cat.
number P7708S). Samples were run under non-reducing and reducing conditions using β-2
mercaptoethanol as reducing agent. Selected gel slices were taken from non-reduced, Coomassie
Blue-stained gels and processed for application to fresh gels under reducing conditions. Samples
were concentrated where required using a Vivaspin 3,000 MWCO PES (Sartorius, Epsom, Surrey, UK)
centrifugal concentrator device operated according to the manufacturer’s instructions.
2.3. Proteomics
Stained protein bands were excised from preparative 1-dimensional gels and analysed by liquid
chromatography-mass spectrometry as previously described [20]. Protein identifications were
assigned using the MASCOT search engine to interrogate protein and gene sequences in the NCBI
databases and the Mustela putorius genome database [21] and linked resources, allowing a mass
tolerance of 0.4 Da for both single and tandem mass spectrometry analyses. BLAST searches, or
searches of genome databases of other Carnivora (e.g. dog and giant panda), were carried out to
check the annotations. Prediction of secretory leader peptides and their cleavage sites were carried
out using SignalP software (http://www.cbs.dtu.dk/services/SignalP/;[22]), and molecular masses
calculated using ProtParam (http://web.expasy.org/protparam/).
3. Results
3.1 Sequential changes in the protein profile of pre-implantation uterine secretions
The protein profiles of all the uterine flush samples collected from days 4 to 14 after mating are
shown in the SDS-PAGE analysis shown in Supplementary Information Figure S1. The disparities
between overall protein concentrations amongst the samples could be due to differences in
efficiency of flushing, changes in the total tissue volume, differences between individual animals’
secretion volumes, or changes in secretory activity. In order to improve comparability, selected
samples were concentrated as described above and/or the volume of loaded sample adjusted to
Proteomics of polecat uterine fluids in early pregnancy
Page 5
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
approximately equalise the intensity of the band at ca. 65 kDa Mr (band C; Figure 1), suspected (and
subsequently confirmed by mass spectrometry), to be serum albumin from its size and slower
migration under reduction.
Standardization of protein concentration to serum albumin revealed dramatic changes in the
chronological protein profile of pregnant uterine flushes, in particular the proteins in bands A, B, D,
E, G, U, and W (Figure 1). The pregnant animals on days 6 and 7 showed few, if any, differences from
the mated but non-pregnant animal at day 4, but by day 9 considerable differences were evident
between pregnant and non-pregnant individuals.
In order to explore relationships between some of the proteins in the most intense bands, gel slices
were excised from a non-reduced gel, and the proteins in them subjected to electrophoresis under
reducing conditions. This showed that bands A and B reduced to a similar set of proteins, suggesting
that they were related or identical (Figure 2). The sizes of the resulting fragments were not
commensurate with post-reduction cleavage products of large multi-chain immunoglobulins (IgM
and IgA) which might be expected to appear in uterine secretions. Protein band C, which was used to
standardize the loading protein concentrations of samples for Figure 1, migrated more slowly when
reduced, which is typical of serum albumin. The change in its migration is usually attributed to
cleavage of its intramolecular disulphide bond such that the unfolded protein is retarded in its
migration. Similar behaviour was exhibited by band G protein, but the others separated as under
non-reducing conditions. The reduction of band C protein uncovered a different protein that was
subjected separately to proteomic analysis (band R).
3.2 Proteomics
Protein bands whose concentrations changed dramatically with time after mating, or whose
concentrations remained constant, were selected for proteomic analysis from both reducing and
non-reducing gels (Figure 1). The separation between the bands in the SDS-PAGE gels was sufficient
to allow proteomic identification of the proteins with high confidence, all directly from the M.
putorius genome-derived protein sequence database. The deductions were unchanged upon
checking by BLAST searching of genomic and other databases for other Carnivora. Table 1 lists the
identifications and also the MASCOT scores and peptide matches of the proteins, and illustrates the
high quality of the matches.
The two bands with the highest Mr, bands A and B, were both identified as α-2-macroglobulin (α2M), a was their reduced form in band J. These sizes are larger than expected from the protein’s
Proteomics of polecat uterine fluids in early pregnancy
Page 6
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
polypeptide mass with its predicted secretory signal peptide removed, and may be due to
glycosylation and/or covalent linkage with other entities. The other proteins that appeared at high
relative amounts around the time of implantation included lipocalin-1 (bands G, U; also known as
tear lipocalin, and an enzyme of the ectonucleotide pyrophosphatase family, also represented in
bands D, O), cathepsin L1 (bands E, W), uteroferrin (bands E, H, W), and zinc-α-2-glycoprotein (band
W). Transferrin (bands F, P), α1-antitrypsin (bands C, R, X), and lactoferrin (bands F, P) remained at
constant levels relative to serum albumin throughout the sampling period.
4. Discussion
The proteins secreted into the uteri of pregnant European polecats changed dramatically in
abundance over the approximately 12 days between mating and the time of implantation by the
embryo. The repertoire of proteins shows some similarities with that found in other groups of
mammals, presumably reflecting a theme inherited from a common ancestor, or convergent
evolution in order to satisfy similar functional requirements. In European polecats, however, the
appearance of α2M was particularly pronounced. Our analysis also identified an abundance of
lipocalin-1, which protein has not been reported as a uterine secretion before.
4.1 α2-macroglobulin
α2M was the most notable protein to appear in abundance shortly before the time of implantation.
It is closely related to pregnancy zone protein (PZP) and three members of the complement system
[23]. It is the largest non-immunoglobulin protein in circulation in mammalian blood, comprises an
approximately 160kDa polypeptide that is glycosylated, and usually occurs in circulation as a
tetramer comprising a pair of non-covalently associated disulphide-linked dimers [23]. It is
synthesised mainly in the liver, but also in the decidua and endometrium in mice and possibly also in
humans, and is thought to be important for implantation [12, 24-27].
α2M is a minor acute-phase inflammation reactant in several species [28]. It operates via two
mechanisms as a broad-spectrum proteinase inhibitor [23]. In one mechanism, an active proteinase
cleaves a bait peptide that causes α2M to fold around and encapsulate the target enzyme. In the
other, the target proteinase is covalently bound via a thioester bond. These mechanisms trap
proteinases of all classes but do not necessarily inactivate them, instead removing them from access
to intact proteins.
Proteomics of polecat uterine fluids in early pregnancy
Page 7
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
In acute-phase and inflammatory responses, the proteinases targeted by α2M are thought to be
those of pathogens, but, significantly, also those released by granulocytes and other immune cells in
potentially damaging inflammatory reactions [23]. This function could be directly relevant to
placentation, given that the sites of placentation in some mammals exhibit local inflammation-like
maternal responses [29-31]. The inflammatory response may be amplified by localized apoptosis of
epithelial cells, and proteinase inhibition by α2M may minimize damage caused by released enzymes
[27, 32-37]. α2M also binds several hormones, growth factors and cytokines involved in
inflammatory responses, and can modify the biological activity of these signalling molecules [19, 23].
In pregnancy, α2M is found in uterine secretions of several species, and has been described to
control trophoblast positioning and overgrowth in the mouse [7-12, 18, 19].
Several bands in the gels of polecat uterine flushes contained α2M, ranging in size from close to that
of the monomeic glycoprotein (band J) to higher-order structures (dimer, tetramer; bands A and B).
Some that did not conform to simple monomers, dimers or tetramers of the protein may be covalent
α2M:proteinase complexes that were not dissembled by the reducing agent (Figure 2). The presence
of α2M in the uterine lumen could be due to increased permeability of the uterine vessels to plasma
proteins or tissue disruption. If plasma leakage were to have contributed to the presence of α2M, as
would also be the case for blood contamination during sampling, then other major plasma proteins,
such as immunoglobulins and complement C3, would be present in proportionately high amounts.
None of these were observed, although serum albumin was clearly present at all times. While this is
the most abundant plasma protein in terms of relative molarity, it is found in uterine secretions of
other species in the absence of other plasma proteins [8, 12]. Moreover, α2M is known to be
synthesised by uterine tissues in humans and mice (see above).
α2M and PZP are closely related proteins. The plasma concentration of PZP increases dramatically in
the course of human pregnancy. Its functions are not understood [38], although it is known to have
proteinase inhibitory activities similar to those of α2M [38, 39]. Despite PZP’s documented synthesis
in several reproductive tissues [38], and that a gene encoding a PZP homologue has been predicted
in the European polecat genome (NCBI accession XP_012906269.1), our analysis identified no
protein that might be identified as PZP in the uterine flushes.
4.2. Lipocalin-1
The appearance of lipocalin-1 was unexpected, as it has not previously been described as a
component of uterine secretions. Moreover, while other lipocalins have been described in the
Proteomics of polecat uterine fluids in early pregnancy
Page 8
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
reproductive tissues of other species, only lipocalin-1 was found in this study of polecat uterine
secretions. Also known as tear lipocalin and von Ebner’s gland protein, lipocalin-1 is a member of a
large family of proteins that operates predominately extracellularly. In addition to its eponymous
presence in tears, lipocalin-1 has been found in a wide range of tissues [40], albeit not in uterine
secretions.
Most lipocalins bind small hydrophobic ligands such as retinol, fatty acids, odorants, or pheromones,
and some are enzymatic [40, 41]. Others are involved in infection defence, such as α1-acid
glycoprotein (orosomucoid), which is an acute-phase protein in humans that binds a range of small
lipophilic molecules and drugs. Lipocalin-2 (neutrophil gelatinase-associated lipocalin) is also an
acute-phase protein and binds bacterial siderophores [42, 43]. Lipocalins associated with
reproduction include progestogen-associated endometrial protein (glycodelin, pregnancyassociated endometrial α-2-globulin, placental protein 14) secreted by the human endometrium
from mid-luteal phase of the menstrual cycle and during the first trimester of pregnancy; a salivary
lipocalin found in pigs is secreted into their uteri at the time of implantation [44]; and equine
uterocalin is secreted by the endometrium of mares in the pre-placentation period, during which the
equine conceptus is confined within a glycoprotein capsule [13, 14]. Despite all these examples of
lipocalins found in reproductive tissues in other species, lipocalin-1 was the only one found in our
European polecat samples.
The biochemical properties of lipocalin-1 are known only from the human form, which binds small
lipids such as fatty acids and sterols [40]. The protein may therefore be involved in delivering or
scavenging lipids at a time when there is significant tissue reorganization and potential for cellular
disruption at implantation, as is speculated for the role of porcine salivary lipocalin [8]. Lipocalin-1,
like lipocalin-2, is known to bind bacterial siderophores, so it may function as part of the innate
immune system in an anti-bacterial role [40, 45]. Human lipocalin-1 also has been recorded as
having cysteinyl proteinase inhibitory activity [46], and nuclease activity that can operate against
viral genomes [47, 48]. The polecat protein may therefore have multiple protective roles in the
uterus, as is thought for its function in tears and epithelia in humans [40, 49].
Taken together, these observations suggest that lipocalin-1 may work alongside α2M to protect the
uterine environment against infections, and its lipid-binding may involve sequestration of toxic lipid
peroxidation products created under condition of oxidative stress [50].
4.3 Tissue-protective and embryo support proteins
Proteomics of polecat uterine fluids in early pregnancy
Page 9
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
In addition to α-2M and lipocalin-1, we identified several additional proteins that may protect the
uterus against infection and tissue damage associated with implantation. These include the ironbinding proteins lactoferrin (lactotransferrin) and transferrin (serotransferrin), both of which were
present at fairly constant levels throughout the sampling period. Lactoferrin in particular is
associated with antimicrobial activity by sequestering iron in milk, it is also synthesised and stored in
neutrophil granules [51], and it directly attacks bacterial membranes and has anti-viral activity [5256].
α1-antitrypsin was also present throughout, and increased in concentration slightly with time. This is
an inhibitor of serine proteinases, is a major acute-phase reactant in humans, and is increasingly
recognized as an anti-inflammatory mediator [57]. Its primary function appears to be protection
against excessive proteolysis of tissues (e.g. in the lower respiratory tract) by neutrophil elastase in
inflammation [58, 59]. It has been observed in pregnant uteri of other species, possibly to control
proteinases released during tissue remodelling, trophoblast invasion, apoptosis, or inflammatory
processes that may accompany placentation [60, 61].
Other proteins increased with time. Cathepsin L1, a cysteinyl proteinase, is usually confined to
lysosomes and is involved in MHC class II antigen processing. Its expression level in uterine tissues is
progesterone-dependent, and its importance in placentation has been recognized [60, 62, 63]. Other
progesterone-dependent proteins that appeared in the polecat uterine fluid included a member of
the ectonucleotide pyrophosphatase family (a lysophospholipase that may be involved in the
production of pharmacologically-active lipid mediators), and uteroferrin (pregnancy-associated acid
phosphatase, which appears to function in transplacental iron transport and stimulation of
erythropoeisis) [16, 64-70].
Curiously, we did not find any dedicated nutrient carrier protein similar to equine uterocalin that
might act to support a developing conceptus during a pre-placentation period. Equine uterocalin
may, however, be a special adaptation in equids to provide resources for a large, capsule-enclosed
conceptus during a prolonged pre-placentation period. Nothing like it has been found in non-equids.
Mustelid blastocysts do have extended placentation periods, and they can even distend the uterus
before they implant, but they have no glycoprotein capsule intervening between their surfaces and
the endometrial surface [6]. Moreover, the surface to volume ratios of mustelid conceptuses are
substantially smaller than in equids at maximum growth such that they may not need a specialized
nutrient carrier. Equine uterocalin and lipocalin-1 bind a similar set of small lipids [15, 40], but the
former is atypically enriched in essential amino acids [15], a feature that is not true of lipocalin-1.
Proteomics of polecat uterine fluids in early pregnancy
Page 10
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
We did, however, find other lipid carriers that may supply the polecat conceptus with lipids, such as
serum albumin and apolipoprotein A-I.
4.4. Conclusion
The protein secretory response of the pre-implantation phase of pregnancy that we observed in
European polecats appears primarily to prevent infection, facilitate implantation, and protect
against release of deleterious cellular components associated with the tissue trauma of
implantation. Some of the lipid-binding proteins we found may also be involved in maternal:embryo
communication by transporting insoluble signalling molecules such as progesterone, prostaglandins
and leukotrienes, or their precursors (as postulated for equine uterocalin; [13, 15]). It would now be
interesting to establish whether species of mustelid that exhibit prolonged obligatory embryonic
diapause (e.g. stoats, otters, badgers) show a similar protein secretory response when reactivation
of blastocysts occurs and implantation is imminent. On the other hand, many of the proteins
identified in this study could fulfil the need to protect and nourish a blastocyst that is free in the
lumen and devoid of circulatory support and direct immune protection for a considerable time
before it implants.
Acknowledgements
We thank Anitta Helin who took care of the polecats, checked on mating success, and assisted with
sample collection, and Suzanne McGill for expert technical assistance with the proteomics analysis.
This study was supported by the University of Eastern Finland, Department of Biosciences, and the
University of Glasgow. Particular thanks go to Dr Kati Loeffler of the International Fund for Animal
Welfare, who critically reviewed the manuscript, found several errors for which the last author was
responsible, and contributed a considerable number of excellent suggestions and changes.
Author contributions
M.W.K. and H.L. conceived, designed, and organised the study. H.L. collected the samples. M.W.K.
and R.J.S.B carried out or oversaw the experiments and analysed the data. M.W.K. wrote the paper
in consultation with the other authors. All authors saw and approved the final submitted version.
Declaration of interests
Proteomics of polecat uterine fluids in early pregnancy
Page 11
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The authors declare no conflicts of interest with respect to the research, authorship and/or
publication of this article.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
References
1.
Wooding, F.B.P. and G.J. Burton, Comparative Placentation: Structures, Functions and
Evolution. 2008, Berlin, Heidelberg: Springer-Verlag.
2.
Ptak, G.E., et al., Embryonic diapause is conserved across mammals. Plos One, 2012. 7(3).
3.
Murphy, B.D., Embryonic diapause: Advances in understanding the enigma of seasonal
delayed implantation. Reproduction in Domestic Animals, 2012. 47: p. 121-124.
4.
Lopes, F.L., J.A. Desmarais, and B.D. Murphy, Embryonic diapause and its regulation.
Reproduction, 2004. 128(6): p. 669-678.
5.
Fenelon, J.C., A. Banerjee, and B.D. Murphy, Embryonic diapause: development on hold.
International Journal of Developmental Biology, 2014. 58(2-4): p. 163-174.
6.
Enders, A.C. and S. Schlafke, Implantation in the ferret: epithelial penetration. American
Journal of Anatomy, 1972. 133(3): p. 291-315.
7.
Ye, T.-M., et al., Two-dimensional liquid chromatography with tandem mass spectrometrybased proteomic characterization of endometrial luminal epithelial surface proteins
responsible for embryo implantation. Fertility and Sterility, 2015. 103(3): p. 853-341.
8.
Kayser, J.P.R., et al., Global characterization of porcine intrauterine proteins during early
pregnancy. Reproduction, 2006. 131(2): p. 379-388.
9.
Smits, K., et al., Proteome of equine oviducal fluid: effects of ovulation and pregnancy.
Reproduction, Fertility and Development, 2016. [Epub ahead of print].
10.
Hayes, M.A., et al., Changes in various endometrial proteins during cloprostenol-induced
failure of early pregnancy in mares. Animal Reproduction, 2012. 9(4): p. 723-741.
11.
Soleilhavoup, C., et al., Proteomes of the female genital tract during the oestrous cycle.
Molecular & Cellular Proteomics, 2016. 15(1): p. 93-108.
12.
Hannan, N.J., et al., 2D-DiGE analysis of the human endometrial secretome reveals
differences between receptive and non-receptive states in fertile and infertile women. Journal
of Proteome Research, 2010. 9(12): p. 6256-6264.
Proteomics of polecat uterine fluids in early pregnancy
Page 12
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
13.
Suire, S., et al., Uterocalin, a lipocalin provisioning the preattachment equine conceptus:
fatty acid and retinol binding properties, and structural characterization. Biochemical
Journal, 2001. 356: p. 369-376.
14.
Stewart, F., M.W. Kennedy, and S. Suire, A novel uterine lipocalin supporting pregnancy in
equids. Cellular and Molecular Life Sciences, 2000. 57(10): p. 1373-1378.
15.
Kennedy, M.W., Uterocalin - provider of essential lipids and amino acids to the preplacentation equine conceptus. Havemeyer Foundation Monograph Series, 2005(16): p. 5356.
16.
Vallet, J.L., R.K. Christenson, and W.J. McGuire, Association between uteroferrin, retinolbinding protein, and transferrin within the uterine and conceptus compartments during
pregnancy in swine. Biology of Reproduction, 1996. 55(5): p. 1172-1178.
17.
Vallet, J.L. and R.K. Christenson, The effect of estrone and estradiol treatment on
endometrial total protein, uteroferrin, and retinol-binding protein secretion during
midpregnancy or midpseudopregnancy in swine. Journal of Animal Science, 1996. 74(11): p.
2765-2772.
18.
Croy, B.A., et al., Update on pathways regulating the activation of uterine Natural Killer cells,
their interactions with decidual spiral arteries and homing of their precursors to the uterus.
Journal of Reproductive Immunology, 2003. 59(2): p. 175-191.
19.
Esadeg, S., et al., Alpha-2 macroglobulin controls trophoblast positioning in mouse
implantation sites. Placenta, 2003. 24(10): p. 912-921.
20.
Griffiths, K., et al., Prolonged transition time between colostrum and mature milk in a bear,
the giant panda, Ailuropoda melanoleuca. Royal Society Open Science, 2015. 2(10).
21.
Peng, X., et al., The draft genome sequence of the ferret (Mustela putorius furo) facilitates
study of human respiratory disease. Nature Biotechnology, 2014. 32(12): p. 1250-U114.
22.
Petersen, T.N., et al., SignalP 4.0: discriminating signal peptides from transmembrane
regions. Nature Methods, 2011. 8(10): p. 785-786.
23.
Rehman, A.A., H. Ahsan, and F.H. Khan, Alpha-2-macroglobulin: A physiological guardian.
Journal of Cellular Physiology, 2013. 228(8): p. 1665-1675.
24.
Bao, L., et al., The role of interleukin-11 in pregnancy involves up-regulation of alpha(2)macroglobulin gene through janus kinase 2-signal transducer and activator of transcription 3
pathway in the decidua. Molecular Endocrinology, 2006. 20(12): p. 3240-3250.
25.
Siu, S.-S.N., et al., Lack of site-specific production of decidual alpha-2 macroglobulin in
human pregnancy. Journal of the Society for Gynecologic Investigation, 2006. 13(7): p. 491496.
Proteomics of polecat uterine fluids in early pregnancy
Page 13
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
26.
Sayegh, R.A., et al., Endometrial alpha-2 macroglobulin; localization by in situ hybridization
and effect on mouse embryo development in vitro. Journal of Clinical Endocrinology &
Metabolism, 1997. 82(12): p. 4189-4195.
27.
Gu, Y., et al., Alpha-2-macroglobulin expression in the mesometrial decidua and its
regulation by decidual luteotropin and prolactin. Endocrinology, 1992. 131(3): p. 1321-1328.
28.
Kuribayashi, T., et al., Elimination half-lives of acute phase proteins in rats and beagle dogs
during acute inflammation. Inflammation, 2015. 38(4): p. 1401-1405.
29.
Dekel, N., et al., The Role of Inflammation for a Successful Implantation. American Journal of
Reproductive Immunology, 2014. 72(2): p. 141-147.
30.
Ott, T.L., et al., Maternal immune responses to conceptus signals during early pregnancy in
ruminants. Animal Reproduction, 2014. 11(3): p. 237-245.
31.
van Mourik, M.S.M., N.S. Macklon, and C.J. Heijnen, Embryonic implantation: cytokines,
adhesion molecules, and immune cells in establishing an implantation environment. Journal
of Leukocyte Biology, 2009. 85(1): p. 4-19.
32.
Siwetz, M., et al., TNF-alpha alters the inflammatory secretion profile of human first
trimester placenta. Laboratory Investigation, 2016. 96(4): p. 428-438.
33.
PrabhuDas, M., et al., Immune mechanisms at the maternal-fetal interface: perspectives and
challenges. Nature Immunology, 2015. 16(4): p. 328-334.
34.
Koets, A.P., The equine endometrial cup reaction - a review. Veterinary Quarterly, 1995.
17(1): p. 21-29.
35.
Noronha, L.E. and D.F. Antczak, Maternal immune responses to trophoblast: The contribution
of the horse to pregnancy immunology. American Journal of Reproductive Immunology,
2010. 64(4): p. 231-244.
36.
Noronha, L.E., et al., Molecular evidence for natural killer-like cells in equine endometrial
cups. Placenta, 2012. 33(5): p. 379-386.
37.
Pampfer, S. and I. Donnay, Apoptosis at the time of embryo implantation in mouse and rat.
Cell Death and Differentiation, 1999. 6(6): p. 533-545.
38.
Tayade, C., et al., Functions of alpha 2 macroglobulins in pregnancy. Molecular and Cellular
Endocrinology, 2005. 245(1-2): p. 60-66.
39.
Chiabrando, G.A., M.A. Vides, and M.C. Sanchez, Differential binding properties of human
pregnancy zone protein- and alpha(2)-macroglobulin-proteinase complexes to low-density
lipoprotein receptor-related protein. Archives of Biochemistry and Biophysics, 2002. 398(1):
p. 73-78.
40.
Dartt, D.A., Tear Lipocalin: Structure and Function. Ocular Surface, 2011. 9(3): p. 126-138.
Proteomics of polecat uterine fluids in early pregnancy
Page 14
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
41.
Flower, D.R., A.C.T. North, and C.E. Sansom, The lipocalin protein family: structural and
sequence overview. Biochimica et Biophysica Acta-Protein Structure and Molecular
Enzymology, 2000. 1482(1-2): p. 9-24.
42.
Flo, T.H., et al., Lipocalin 2 mediates an innate immune response to bacterial infection by
sequestrating iron. Nature, 2004. 432(7019): p. 917-921.
43.
Berger, T., et al., Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli
infection but not to ischemia-reperfusion injury. Proceedings of the National Academy of
Sciences of the United States of America, 2006. 103(6): p. 1834-1839.
44.
Seo, H., et al., Salivary lipocalin is uniquely expressed in the uterine endometrial glands at the
time of conceptus implantation and induced by interleukin-1beta in pigs. Biology of
Reproduction, 2011. 84(2): p. 279-287.
45.
Fluckinger, M., et al., Human tear lipocalin exhibits antimicrobial activity by scavenging
microbial siderophores. Antimicrobial Agents and Chemotherapy, 2004. 48(9): p. 3367-3372.
46.
Hof, W.v.t., et al., The salivary lipocalin von Ebner's gland protein is a cysteine proteinase
inhibitor. Journal of Biological Chemistry, 1997. 272(3): p. 1837-1841.
47.
Yusifov, T.N., et al., Endonuclease activity in lipocalins. Biochemical Journal, 2000. 347: p.
815-819.
48.
Yusifov, T.N., et al., Tear lipocalin is the major endonuclease in tears. Molecular Vision, 2008.
14(19-23): p. 180-188.
49.
Redl, B., et al., Identification of a lipocalin in mucosal glands of the human tracheobronchial
tree and its enhanced secretion in cystic fibrosis. Laboratory Investigation, 1998. 78(9): p.
1121-1129.
50.
Lechner, M., P. Wojnar, and B. Redl, Human tear lipocalin acts as an oxidative-stress-induced
scavenger of potentially harmful lipid peroxidation products in a cell culture system.
Biochemical Journal, 2001. 356(1): p. 129-135.
51.
Martin, L., et al., Antimicrobial peptides in human sepsis. Frontiers in Immunology, 2015. 6.
52.
Brouwer, C.P.J.M., M. Rahman, and M.M. Welling, Discovery and development of a synthetic
peptide derived from lactoferrin for clinical use. Peptides, 2011. 32(9): p. 1953-1963.
53.
Wada, Y. and B. Lönnerdal, Bioactive peptides derived from human milk proteins —
mechanisms of action. The Journal of Nutritional Biochemistry, 2014. 25(5): p. 503-514.
54.
Vogel, H.J., Lactoferrin, a bird’s eye view. Biochemistry and Cell Biology, 2012. 90(3): p. 233244.
55.
Abril Garcia-Montoya, I., et al., Lactoferrin a multiple bioactive protein: An overview.
Biochimica et Biophysica Acta-General Subjects, 2012. 1820(3): p. 226-236.
Proteomics of polecat uterine fluids in early pregnancy
Page 15
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
56.
Valenti, P. and G. Antonini, Lactoferrin: an important host defence against microbial and
viral attack. Cellular and Molecular Life Sciences, 2005. 62(22): p. 2576-2587.
57.
Bergin, D.A., et al., The circulating proteinase inhibitor alpha-1 antitrypsin regulates
neutrophil degranulation and autoimmunity. Science Translational Medicine, 2014. 6(217).
58.
McElvaney, N.G., et al., Aerosol alpha-1-antitrypsin treatment for cystic-fibrosis. Lancet,
1991. 337(8738): p. 392-394.
59.
Birrer, P., et al., Protease-antiprotease imbalance in the lungs of children with cystic-fibrosis.
American Journal of Respiratory and Critical Care Medicine, 1994. 150(1): p. 207-213.
60.
Li, W.G., et al., Progesterone-dependent cathepsin-l proteolytic activity in cat uterine
flushings. Biology of Reproduction, 1991. 44(4): p. 625-631.
61.
Geisert, R.D., et al., Characterization and proteolytic activity of a cathepsin L-like polypeptide
in endometrium and uterine flushings of cycling, pregnant and steroid-treated
ovariectomized gilts. Reproduction Fertility and Development, 1997. 9(4): p. 395-402.
62.
Varanou, A., et al., The importance of cysteine cathepsin proteases for placental
development. Journal of Molecular Medicine-Jmm, 2006. 84(4): p. 305-317.
63.
Screen, M., et al., Cathepsin proteases have distinct roles in trophoblast function and
vascular remodelling. Development, 2008. 135(19): p. 3311-3320.
64.
Ahn, H.J. and E.B. Jeung, Distinct expression and regulation of ectonucleotide
pyrophosphatase/phosphodiestrase 2 and its potential role(s) in the uterus of rats during
estrous cycle. Reproduction Fertility and Development, 2011. 23(1): p. 191-191.
65.
Ahn, H.-J. and E.-B. Jeung, The expression of ENPP2 in rat uterus during the estrous cycle.
Biology of Reproduction, 2010: p. 123-123.
66.
Ahn, H.-J., et al., Expression and regulation of Enpp2 in rat uterus during the estrous cycle.
Journal of Veterinary Science, 2011. 12(4): p. 379-385.
67.
Hood, B.L., et al., Proteomics of the human endometrial glandular epithelium and stroma
from the proliferative and secretory phases of the menstrual cycle. Biology of Reproduction,
2015. 92(4).
68.
Carter, A.M. and A.C. Enders, The evolution of epitheliochorial placentation, in Annual
Review of Animal Biosciences, Vol 1, H.A. Lewin and R.M. Roberts, Editors. 2013. p. 443-467.
69.
Ellenberger, C., et al., Immunolocalisation of the uterine secretory proteins uterocalin,
uteroferrin and uteroglobin in the mare's uterus and placenta throughout pregnancy.
Theriogenology, 2008. 70(5): p. 746-757.
70.
Ying, W., et al., Pregnancy-secreted acid phosphatase, uteroferrin, enhances fetal
erythropoiesis. Endocrinology, 2014. 155(11): p. 4521-4530.
Proteomics of polecat uterine fluids in early pregnancy
Page 16
Table 1. Identification of the proteins isolated from bands excised from the protein electrophoresis gels indicated in Figures 1 and 2.
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
A, B,
α2-macroglobulin
H, I, J,
163781.5
XP_004779762
3502
215 (127)
triggers enclosure of target proteinase. Thio-ester bond-
(161296.4)
K, M,
Inhibitor of all classes of proteinase. Bait and trap mechanism
mediated covalent binding of proteinases. Largest major nonimmunoglobulin protein complex in plasma. Acute-phase
O
protein in rats, minor acute phase reactant in other species.
Elevated in pregnancy in humans and mice. Controls foetal
positioning. Functionally distinct isoforms.
C, R,
X
α1-antitrypsin
46521.2
XP_004754815
663
51 (26)
Serine proteinase inhibitor. Acute-phase reactant.
(44096.2)
Proteomics of polecat uterine fluids in early pregnancy
Page 17
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
G, U
lipocalin-1
19342.0
XP_004757035
317
23 (11)
Bacterial siderophore-binding. Endonuclease. Cystatin –
(17491.7)
W
Zinc-α-2glycoprotein
C
Serum albumin
36407.5
cysteinyl proteinase inhibitor.
XP_004781894
310
22(9)
Lipid metabolism signal; adipokine.
XP_004766346
1425
99 (52)
Carrier of fatty acids and other small lipids, other small
(34360.9)
68597.4
molecules and drugs, osmotic regulation in circulatory
(66474.9)
F, P
Serotransferrin
Syn. tear lipocalin, von Ebner’s gland protein. Lipid transport.
78378.1
system.
XP_004762537
849
59 (27)
Syn. transferrin. Iron transporter.
(76408.6)
Proteomics of polecat uterine fluids in early pregnancy
Page 18
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
F, P
Lactotransferrin
77327.0
XP_004761226
509
37 (20)
regulator in inflammation. Synthesised and stored in
(75354.4)
E, W
Cathepsin L1
37047.3
neutrophil granules.
XP_004782389
343
34 (17)
W
Uteroferrin
37645.9
Cysteinyl proteinase. Lysosomal. Antigen processing in MHC II
pathway. Extracellular matrix modification. Endometrial
(35338.2)
E, H,
Syn. lactoferrin. Iron capture, antimicrobial, negative
remodelling.
XP_004748428
(35353.2)
464
24 (13)
Tartrate-resistant acid phosphatase type 5 (ACP5).
Pregnancy-associated acid phosphatase. Synthesized in
response to progesterone. Widely conserved uterine protein
in mammals. Appears to function in transplacental iron
transport and stimulation of erythropoeisis. Human – bone
building & remodelling, negative regulator of inflammatory
signals.
Proteomics of polecat uterine fluids in early pregnancy
Page 19
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
D, O
Ectonucleotide
pyrophosphatase
R
Legumain
109707.5
XP_004743546
443
60 (19)
Appears to modulate insulin sensitivity and function; insulin
(none)
49280.8
receptor binding.
XP_004739089
276
16 (7)
Haemoglobin β
15994.3
Hydrolyses proteins at -Asn-Xaa- site. Multifunctional.
Processing of proteins for MHC class II antigen presentation
(47556.7)
T
Ectoenzyme. Bone mineralization and soft tissue calcification.
in the lysosomal/endosomal system.
XP_004779082
430
24 (13)
O2/CO2 transport and exchange.
(none)
Proteomics of polecat uterine fluids in early pregnancy
Page 20
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
D, O
Galectin-3binding protein
62371.8
XP_004748843
281
22 (9)
Galactose-specific lectin required for terminal differentiation
of columnar epithelial cells during early embryogenesis;
(60418.3)
involved in acute inflammatory responses. Promotes
intergrin-mediated cell adhesion. May stimulate host defence
against viruses and tumour cells.
D, O
S
Complement C2
Apolipoprotein
A-I
82486.9
267
(80560.4)
XP_012905650
30179.2
XP_004749957
(28334.9)
Proteomics of polecat uterine fluids in early pregnancy
23 (11)
Component of the complement system. Cell lysis.
Inflammation.
458
31 (13)
Lipid binding. Cholesterol transport. Major protein
component of high density lipoprotein (HDL) in plasma.
Constituent of milk.
Page 21
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
Alkaline
I
phosphatase,
tissue-
57449.3
XP_004741321
135
12 (3)
Non-specific phosphomonoesterases possibly involved in cell
signalling and bone mineralisation. Enzymes of this class
(55717.2)
found widely in liver, bile duct, kidney, bone, intestinal
nonspecific
mucosa and placenta.
isozyme isoform
X1
Aminopeptidase
I
110639.6
XP_012917463
127
12 (4)
Broad spectrum aminopeptidase. May be involved in the
metabolism of regulatory peptides of diverse cell types.
N
Responsible for the processing of peptide hormones.
M
IgG Fc-binding
234543.3
XP_012918171
protein
401
57 (16)
Binds Fc fragment of immunoglobulin G (IgG). May be
involved in the maintenance of mucosal structure as a gellike component of the mucosa.
Proteomics of polecat uterine fluids in early pregnancy
Page 22
Banda
Protein b
Mass of
Database
MASCOT Number of
protein c
accession code d
score e
Function, association, synonyms and comments g
peptides
(unique
peptide
matches) f
X
Cathepsin D
44455.3
XP_004759751
718
54 (25)
Oestrogen-regulated transcript in breast cancer cells.
(42483.8)
X
Uteroferrinassociated basic
protein 2
48617.4
Acid protease active in intracellular protein breakdown.
XP_004754814
307
68 (17)
Serine proteinase inhibitor.
(45884.0)
a
Gel band codes as indicated in Figures 1 and 2.
b
Protein identifications. Peptides matching to keratin were excluded.
c
The predicted molecular masses, as calculated by ProtParam (http://web.expasy.org/protparam/), are of the complete polypeptides encoded before
removal of any leader/signal peptides for secretion. The predicted masses following removal of such leader peptides at positions predicted by Signalp is
listed in brackets ([22]. Note posttranslational modifications, principally glycosylation, will alter mobility in protein electrophoresis gels.
d
Accession number/reference sequence from NCBI Genbank database for Mustela putorius genome database and checked by BLAST searching.
Proteomics of polecat uterine fluids in early pregnancy
Page 23
e
MASCOT (MOWSE) search score where scores greater than 38 are taken to be significant. The MASCOT score is the highest value obtained where the
protein was identified in more than one band, as were the peptide match values.
f
Number of peptides found to match with number of peptides unique to this identification in parentheses.
g
Putative functions and comments are drawn from literature cited, or NCBI and UniProtKB/Swiss-Prot databases.
Proteomics of polecat uterine fluids in early pregnancy
Page 24
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Legends to Figures
Figure 1. Changes in European polecat uterine secretory proteins with time after mating. Animals
sampled on days 4 and 14 were non-pregnant so act as controls. The sample volumes were adjusted
to normalize the intensity of the strong band at approximately 65 kDa (serum albumin) among
tracks. See Figure S1 for SDS-PAGE of all of the samples collected and upon which the adjustments
were based. Gel band codes indicated by letters and are referred to in the text and in Table 1. M –
marker/calibration proteins with relative mobilities (Mr) as indicated in kilodaltons (kDa).
Figure 2. Subunit composition of major proteins in European polecat uterine flush following
protein reduction. The labelled bands were excised from a non-reducing preparative SDS-PAGE gel
of a uterine flush sample taken from a pregnant animal on day 12 after mating and re-run under
reducing conditions. The letter codes for each band are consistent with those in Figure 1. Protein
samples were run under reducing or non-reducing (NR) conditions. M – marker/calibration proteins
with relative mobilities (Mr) indicated in kilodaltons (kDa).
Proteomics of polecat uterine fluids in early pregnancy
Page 25
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 1.
Proteomics of polecat uterine fluids in early pregnancy
Page 26
bioRxiv preprint first posted online Nov. 18, 2016; doi: http://dx.doi.org/10.1101/088609. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2.
Proteomics of polecat uterine fluids in early pregnancy
Page 27